The Metabolomics Core (B) is comprised of a Biosafety Level 3 suite for handling infectious samples, massively parallel detection of mycobacterial metabolites using Time of Flight mass spectrometry (Agilent Accurate Mass ToF 6230), specialized resources for identifying known mycobacterial compounds, and analytical capabilities to discover previously unknown compounds (Agilent Accurate Mass 6520, QTof, Thermo LXQ Advantage 2 Dimensional Ion Trap with MSn). The integrated technologies were assembled by the Moody laboratory and supported a Biomarker Discover Initiative of the Broad Institute, NIH U19 and ROl Projects. Dataflow involves receipt of mycobacteria (Project 3) or patient samples (Project 4), from which total metabolites are extracted and sterilized in organic solvents. Tissue or mycobacterial extracts enter into a liquid chromatography-mass spectrometry system, which was specifically designed to broadly detect the highly diverse and hydrophobic compounds in mycobacteria. In the first phase of whole organism analysis, the platform rapidly detects triplicate intensity values for ~10,000 distinct compounds in each sample. By aligning large datasets derived from different patients, clinical isolates or genetically engineered bacteria, in-house-designed software pipeline identifies all compounds that are changed at statistically significant levels. In a second, targeted phase, all changed compounds are ranked by biological or quantitative criteria to define compounds of interest, whose structures are solved by comparing their masses to the literature (MycoMass) and in-house (MycoMap) databases or are solved through collisional mass spectrometry. This system has discovered previously unknown compounds, identified strain-specific mycobacterial biomarkers in vitro and from tissues and identified lipids changed after gene deletion. This overview describes expansion of the substantial existing core facilities, including a new generation of high accuracy mass spectrometry and expansion of mycobacterial databases, as well as use of the Core to discover biomarkers in drug-resistant or latent mycobacteria or biomarkers of infection.